Vaccines comprise antigens or combinations of antigens which when administered to a warm-blooded animal prevent, ameliorate or treat disease. Vaccines for.infectious diseases originally comprised whole, attenuated or killed microbes. It was soon discovered however that only a few proteins or protein fragments of a microbe or cell stimulated a protective immune response, and, in fact, inclusion of extraneous materials from the whole cell could hinder the immune response. Therefore, vaccine development focused on identifying the particular protein, protein fragment, epitope and DNA segment encoding that epitope which elicited the protective immune response. As antigen identification became more precise however, vaccine efficiency declined. Identified: antigens were often small molecules unable to be recognized by antigen processing cells. It was therefore necessary to combine these antigens with substances which enhance the antigenicity of the antigen and give a superior immune response. These substances are adjuvants.
Adjuvants work by several means. Some assist in the presentation of antigen to antigen processing cells (APC). Oil-in-water emulsions, water-in-oil emulsions, liposomes and microbeads each assist in presenting antigen to APC. Small antigens or haptens are often linked to larger, immunogenic proteins or polysaccharides to facilitate recognition by the APC. Certain adjuvants have a depot effect holding antigen in place until the body has an opportunity to mount an immune response. Other adjuvants stimulate the immune system generally augmenting the specific response mounted to the antigen.
The attenuated lipid A derivatives (ALD) monophosphoryl lipid A (MLA) and 3-deacylated monophosphoryl lipid A (3D-MLA) are potent immunological adjuvants used in prophylactic vaccines for infectious disease and therapeutic vaccines for the treatment of cancerous tumors and chronic infections. MLA and 3D-MLA are modified forms of the bacterial endotoxin lipopolysaccharide (LPS) and are known and described in U.S. Pat. Nos. 4,436,727 and 4,912,094, respectively. MLA and 3D-MLA induce both a humoral antibody response and a cell-mediated immune response in patients administered the compounds with an antigen.
An effective vaccine presents antigens to a warm-blooded animal such that the animal can mount a protective immune response to those antigens. Often, a vaccine composition must include an adjuvant to achieve this effect. Adjuvants which stimulate both a humoral and cellular immune response and are safe and non-toxic would promote the efficacy of any vaccine.
The subject invention is a novel adjuvant composition. The adjuvant composition is a stable oil-in-water emulsion (SE) comprising a metabolizable oil, surfactants, an antioxidant and a component to make the emulsion isotonic. The particle size of the claimed stable emulsion is less than 130 nm to 3 xcexcm. Emulsions in the range of 70-200 nm can be sterilized by filtration. The hydrophobic-lipophilic balance (HLB) of the stable emulsion.is from about 7.5 to about 10.5 and preferably about 8.0.
In a preferred embodiment, the adjuvant composition is combined with an attenuated lipid A derivative (ALD). The addition of an ALD increases the adjuvanticity of the composition. ALDs useful according to the subject invention include monophosphoryl lipid A and 3-deacylated monophosphoryl lipid A. ALD can be included in the formulation at a concentration ranging from about 1 xcexcg-12,000 xcexcg/ml. Vaccine compositions of the novel stable emulsion are also claimed.
The subject invention is an adjuvant composition which is a stable oil-in-water emulsion comprising a metabolizable oil, surfactants, an antioxidant and a component to make the emulsion isotonic. The resulting emulsion is buffered, has a particle size of less than 3 xcexcm and a hydrophobic-lipophilic balance of the stable emulsion is from about 7.5 to about 10.5 and preferably about 8.0.
In a preferred embodiment the stable emulsion comprises from about 2% to about 15%, and preferably 10%, volume/volume of the metabolizable oil squalene. Surfactants are present in the stable emulsion at about 2%. Approximately 50 xcexcg of an antioxidant can be added to the stable emulsion of the subject invention and approximately 1.75% of an agent to make the emulsion isotonic.
Meabolizable oils useful according to the subject invention include squalene, soybean oil, sesame oil and caprylic/capric acid triglycerides (MIGLYCOL 810 oil). Squalene is preferred.
Surfactants useful according to the subject invention are Tween 80, polysorbate 80 (CAPMUL POE-O low PV surfactant, ABITEC Corp., Janesville, Wis.), polyethylene 660 12-hydroxystearate (SOLUTOL HS15, BASF Corp., Chicago, Ill.) and poloxamer 188 (PLURONIC Q F68 block co-polymer, BASF Corp., Chicago, Ill.), sodium cholate, glycerodeoxy cholate, phosphatidyl choline, with poloxamer 188 being preferred. It was found that Tween 80 and polysorbate 80 surfactant produced a histamine type response when administered intravenously to dogs. Other suitable surfactants include sphingolipids such as sphingomyelin and sphingosine and phospholipids such as egg phosphatidylcholine, 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, L-xcex1-Phosphatidylethanolamine, and 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixtures thereof. DPPC is acceptable for use in humans.
Antioxidants useful in the stable emulsion of the subject invention include a tocopherol, and ascorbic acid, with xcex1 tocopherol being preferred.
Agents that can be added to the emulsion of the subject invention to make the adjuvant isotonic include dextrose, glycerol, mannitol, sorbitol, PEG 300, PEG 400 and polyethylene glycol, with glycerol being preferred.
In a particularly preferred embodiment, an attenuated lipid A derivative (ALD) is incorporated into the compositions of the subject invention. ALDs are lipid A-like molecules that have been altered or constructed so that the molecule displays lesser or different of the adverse effects of lipid A. These adverse effects include pyrogenicity, local Shwarzman reactivity and toxicity as evaluated in the chick embryo 50% lethal dose assay (CELD50) ALDs useful according to the subject invention include monophosphoryl lipid A (MLA) and 3-deacylated monophosphoryl lipid A (3D-7MLA). MLA and 3D-MLA are known and need not be described in detail herein. See for example U.S. Pat. No. 4,436,727 issued Mar. 13, 1984, assigned to Ribi ImmunoChem Research, Inc., which discloses monophosphoryl lipid A and its manufacture. U.S. Pat. No. 4,912,094 and reexamination certificate B1 U.S. Pat. No. 4,912,094 to Myers, et al., also assigned to Ribi ImmunoChem Research, Inc., embodies 3-deacylated monophosphoryl lipid A and a method for its manufacture. Disclosures of each of these patents with respect to MLA and 3D-MLA are incorporated herein by reference.
Without going into the details of the prior incorporated by reference patents, monophosphoryl lipid A (MLA) as used herein is derived from lipid A, a component of enterobacterial lipopolysaccharides (LPS), a potent but highly toxic immune system modulator. Edgar Ribi and his associates achieved the production of monophosphoryl lipid A (MLA) referred to originally as refined detoxified endotoxin (RDE). MLA is produced by refluxing an endotoxin extract (LPS or lipid A) obtained from heptoseless mutants of gram-negative bacteria in mineral acid solutions of moderate strength (0:1 N HCl) for a period of approximately 30 minutes. This treatment results in the loss of the phosphate moiety at position 1 of the reducing end glucosamine.
Coincidentally, the core carbohydrate is removed from the 6 position of the non-reducing glucosamine during this treatment. The resulting product (MLA) exhibits considerable attenuated levels of the endotoxic activities normally associated with the endotoxin starting material, such as pyrogenicity, local Shwarzman reactivity, and toxicity as evaluated in the chick embryo 50% lethal dose assay (CELD50). However, it unexpectedly retains the functionality of lipid A and LPS as an immunomodulator.
Another detoxified endotoxin which may be utilized in the practice of the present invention is referred to as 3-deacylated monophosphoryl lipid A (3D-MLA). 3D-MLA is known as set forth in U.S. Pat. No. 4,912,094, reexamination certificate B1 U.S. Pat. No. 4,912,094, and differs from MLA in that there is selectively removed from the MLA molecule the B-hydroxymyristic acyl residue that is ester linked to the reducing-end glucosamine at position 3 under conditions that do not adversely affect the other groups. 3-deacylated monophosphoryl lipid A is available from Ribi ImmunoChem Research, Inc., Hamilton, Mont. 59840.
The MLA and 3D-MLA molecules are a composite or mixture of a number of fatty acid substitution patterns, i.e., heptaacyl, hexaacyl, pentaacyl, etc., with varying fatty acid chain lengths. Thus, various forms of MLA and 3D-MLA, including mixtures thereof, are encompassed by this invention. The lipid A backbone that is illustrated in U.S. Pat. No. 4,912,094 and reexamination certificate B1 U.S. Pat. No. 4,912,094 corresponds to the product that is obtained by 3-deacylation of heptaacyl lipid A from S. Minnesota R595. Other fatty acid substitution patterns are encompassed by this disclosure; the essential feature is that the material be 3-deacylated.
The modified 3D-MLA utilized in the present invention is prepared by subjecting MLA to alkaline hydrolysis under conditions that result in the loss of but a single fatty acid from position 3 of the lipid A backbone. xcex2-hydroxymyristic fatty acid at position 3 is unusually labile in alkaline media. It requires only very mild alkaline treatment to completely 3-deacylate lipid A. The other ester linkages in lipid A require somewhat stronger conditions before hydrolysis will occur so that it is possible to selectively deacylate these materials at position 3 without significantly affecting the rest of the molecule. The reason for the unusual sensitivity to alkaline media of the ester-linked xcex2-hydroxymyristic fatty acid at position 3 is not known at this time.
Although alkaline hydrolysis procedures are known, it is important to choose conditions that do not cause further hydrolysis beyond the ester linkage to the xcex2-hydroxymyristic at position 3.
In general the hydrolysis can be carried out in aqueous or organic media. In the latter case, solvents include methanol (alcohols), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), chloroform, dichloromethane, and the like, as well as mixtures thereof Combinations of water and one or more of the mentioned organic solvents also can be employed.
The alkaline base can be chosen from among various hydroxides, carbonates, phosphates and amine. Illustrative bases include the inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and the like, and organic bases such as alkyl amines, and include, but are not limited to, diethylamine, triethylamine, and the like.
In aqueous media the pH is typically between approximately 10 and 14 with a pH of about 12 to about 13.5 being the preferred range. The hydrolysis reaction is typically carried out at a temperature of from about 20xc2x0 C. to about 80xc2x0 C., preferably about 50xc2x0 C. to 60xc2x0 C. for a period of about 10 to about 30 minutes. For example, the hydrolysis can be conducted in 3% triethylamine in water at room temperature (22xc2x0-25xc2x0 C.) for a period of 48 hours. The only requirement in the choice of temperature and time of hydrolysis is that deacylation occurs to remove only the xcex1-hydroxymyristic at position 3.
In practice it has been found that a particularly desirable hydrolysis method involves dissolving lipid A or monophosphoryl lipid A in chloroform:methanol 2:1 (v/v), saturating this solution with an aqueous buffer consisting of 0.5M Na2CO3 at pH 10.5, and then flash evaporating the solvent at 45xc2x0-50xc2x0 C. under a vacuum for an aspirator (approximately 100 mm Hg). The resulting material is selectively deacylated at position 3. This process can also be carried out with any of the inorganic bases listed above. The addition of a phase transfer catalyst, such as tetrabutyl ammonium bromide, to the organic solution prior to saturation with the aqueous buffer may be desirable in some cases. In addition to MLA and 3D-MLA produced as described above, ALD produced by synthetic or semi-synthetic processes may be used.
The composition of the subject invention is an adjuvant. When an effective amount of the composition is administered to a host with a protein antigen. The host""s immune response to that antigen is enhanced. An effective amount of the claimed adjuvant composition is a quantity which stimulates or enhances an immune response. One skilled in the art would know the amount of antigen which is necessary to stimulate an immune response to that antigen. For example, 2.5 xcexcg of hepatitis B surface antigen (HBsAg) administered with a preferred embodiment of the subject invention induced a humoral response in mice.
It has been unexpectedly found that the stable emulsion of the subject invention when combined with an ALD significantly reduces the pyrogenicity of the ALD. Pyrogenicity is the production of a febrile state by a compound. The ALD, 3D-MLA produces a higher febrile response when formulated in 40% polyethylene glycol, 10% ethanol than when formulated in the stable emulsion of the subject invention. Pyrogenicity of a composition can be evaluated in a standard three rabbit USP pyrogen test. Briefly, three rabbits are administered the compounds at varying doses. Each animal""s body temperature is monitored over the course of 4 hours. Any temperature decrease is recorded as a rise of zero. An individual rise in temperature of less than 0.5xc2x0 F. was considered non-pyrogenic. If the composition causes an individual rise in temperature of 0.5xc2x0 F. or more, the composition is retested using five different rabbits. If not more than three of the eight total rabbits exhibited a rise in temperature of 0.5xc2x0 F. or more and if the sum of the rise in temperature for each of the eight rabbits does not exceed 3.3xc2x0 F., the composition is considered non-pyrogenic.